PROJECT SUMMARY The Rb tumor suppressor is mutationally inactivated in relatively small fraction of lung adenocarcinomas, but the Rb pathway in general is likely inactivated in the majority of cases. This scenario sets the stage for therapies targeting the upstream negative regulators of Rb, namely cyclin dependent kinases, to reactivate Rb's tumor suppressive functions. Currently, multiple clinical trials are underway to test the efficacy of such therapies and significant clinical benefits have been discovered in certain cancer types. However, even in these cases the therapeutic durability is uncertain and it is currently unknown whether Rb reactivation will be effective in many other tumor types including lung adenocarcinoma. Adding to this uncertainty is the surprising realization that the role of Rb, or the Rb pathway in general, in oncogenic Kras- driven lung adenocarcinomas is unclear. Therefore, our project has two major goals: Aim 1 focuses on determining the functional consequences of Rb mutations in both genetically engineered mouse lung adenocarcinoma models and in human lung cancer specimens. In contrast, Aim 2 of this proposal focuses on modeling Rb reactivation therapy using a genetic tool recently developed in our laboratory that allows both, the conditional inactivation of Rb during tumor development, and the inducible and accurate reactivation of Rb once cancers are established. Fueled by our preliminary findings, our overarching hypothesis in Aim 1 is that loss of Rb accelerates lung adenocarcinoma progression by removing two distinct barriers that work in sequence to limit the adenoma-carcinoma transition, and then the onset of metastatic competency. In Aim 2, our initial insights suggest the hypothesis that the major role of Rb restoration in established lung adenocarcinomas is to reestablish repressive chromatin structures that lead to the reversal of advanced tumor grades and repression of pro-metastatic gene expression programs. We expect that our study will uncover the mechanisms that drive selection of Rb pathway mutations, and establish the therapeutic efficacy of Rb pathway restoration in lung adenocarcinoma. Further, our study will highlight specific biochemical programs utilized by the Rb pathway in disease relevant contexts that could be therapeutically stimulated to recapitulate the natural functions of this critical tumor suppressor. These insights may be broadly applicable to the many tumor types that harbor Rb pathway mutations.